Electrical signaling in bacterial biofilms Grol Sel (P.I.), Lev Tsimring (Co-I.) and Andrew Mugler (Co-I.) Summary Understanding communication among bacteria is a fundamental biological problem with critical implications for public health. Nowhere is this problem perhaps more relevant than in bacterial communities known as biofilms. How individual bacteria communicate within such collectively organized communities to coordinate their behavior is a long-standing question in biology. We recently discovered a new type of bacterial communication mechanism based on ion channel mediated electrical cell-to-cell signaling in Bacillus subtilis biofilms. This discovery made possible by our recent technical innovations, ideally positions us to tackle the fundamental and long-standing question regarding how bacteria coordinate their behavior in biofilms. Necessitated by the multi- scale nature of the problem, the specific aims we propose are designed to bridge across spatial and temporal scales: 1) How does cell density and heterogeneity influence long-range communication within the biofilm? We propose that high cell density and cell-to-cell heterogeneity in electrical activity promotes efficient signal transmission within the biofilm. 2) What governs the composition and dispersion of biofilm communities? We propose that long-range electrical signaling plays a critical role in attracting individual bacteria to, or repelling them from the biofilm. 3) Does a biofilm operate as an independent functional unit in the presence of neighboring biofilms? We propose that multiple neighboring biofilms can become coupled through long-range electrical signaling into a super-cluster that can act as a functional unit. These questions involve phenomena operating over length scales ranging from individual cells to groups of biofilms, and timescales ranging from seconds to hours. Therefore, multi-scale quantitative time-lapse microscopy is the essential tool to address these questions. Importantly, the resulting quantitative spatio-temporal measurements are ideal to inform and constrain problem specific mathematical models and determine the role of electrical signaling in coordinating bacteria in biofilms. The quantitative spatio-temporal measurements to be generated here will thus also serve the broad interests of the computational biology community. !